Antimicrobial proteins from Allium

ABSTRACT

PCT No. PCT/GB94/01636 Sec. 371 Date Jan. 25, 1996 Sec. 102(e) Date Jan. 25, 1996 PCT Filed Jul. 29, 1994 PCT Pub. No. WO95/04754 PCT Pub. Date Feb. 16, 1995Antimicrobial proteins capable of isolation from seeds of Allium show a wide range of antifungal activity and some activity against Gram-positive bacteria. DNA encoding the proteins may be isolated and incorporated into vectors. Plants transformed with this DNA may be produced. The proteins find commercial application as antifungal or antibacterial agents; transformed plants will show increased disease resistance.

This application claims benefit of international applicationPCT/GB94/01636, filed Jul. 29, 1994.

This invention relates to antimicrobial proteins, processes for theirmanufacture and use, and DNA sequences coding for them.

In this context, antimicrobial proteins are defined as proteinspossessing at least one of the following activities: antifungal activity(which may include anti-yeast activity); antibacterial activity.Activity includes a range of antagonistic effects such as partialinhibition or death. Such proteins may be oligomeric or may be singlepeptide subunits.

Various proteins with antimicrobial activity have been isolated fromplant sources, and such proteins are often believed to take part in hostdefence mechanisms directed against invading or competingmicro-organisms. Some of the proteins are well-characterised, and theiramino acid sequence may be known. In some cases, the cDNA or geneencoding the protein has also been isolated and sequenced.

To keep out potential invaders, plants produce a wide array ofantifungal compounds, either in a constitutive or an inducible manner.Several classes of proteins with antifungal properties have now beenidentified, including:

chitinases (Schlumbaum A et al, 1986, Nature, 324, 363-367);

beta-1,3-glucanases (Mauch F et al, 1988, Plant Physiol, 88, 936-942);

chitin-binding lectins (Broekaert WF et al, 1989, Science, 245,1100-1102; Van Parijs J et al, 1991, Planta, 183, 258-264);

permatins (including zeamatins) (Roberts WK and Selitrennikoff CP, 1990,J Gen Microbiol, 136, 2150-2155; Vigers AJ et al, 1991, MolecPlant-Microbe Interact, 4, 315-323; Woloshuk CP et al, 1991, Plant Cell,3, 619-628);

thionins (Bohlmann and Apel, 1991, Ann Rev Plant Physiol Plant Mol Biol,42:227-240);

ribosome-inactivating proteins (Roberts WK and Selitrennikoff CP, 1986,Biosci Rep, 6, 19-29; Leah et al, 1991, J Biol Chem, 266, 1564-1573;Carrasco et al, 1981, Eur J Biochem, 116, 185-189; Vernon et al, 1985,Arch Biochem Biophys, 238, 18-29; Stirpe and Barbieri, 1986, FEBS Lett,195, 1˜8).

These proteins have gained considerable attention as they couldpotentially be used as biocontrol agents.

Other groups of antimicrobial proteins with activity against plantpathogenic fungi (and often some antibacterial activity) are capable ofisolation from certain plant species. We have previously described thestructural and antifungal properties of several such proteins,including:

the small-sized cysteine-rich proteins Mj-AMP1 (antimicrobial protein 1)and Mj-AMP2 occurring in seeds of Mirabilis jalapa (Cammue BPA et al,1992, J Biol Chem, 267:2228-2233; International Application PublicationNumber WO92/15691 published on 17 Sep. 1992);

Ac-AMP1 and Ac-AMP2 from Amaranthus caudatus seeds (Broekaert WF et al,1992, Biochemistry, 37:4308-4314; International Application PublicationNumber WO92/21699 published on 10 Dec. 1992);

Ca-AMP1 from Capsicum annuum, Bm-AMP1 from Briza maxima and relatedproteins found in other plants including Delphinium, Catapodium,Baptisia and Microsensis species (International Patent ApplicationPublication Number WO94/11511, published 26 May 1994);

Rs-AFP1 (antifungal protein 1) and Rs-AFP2 from seeds of Raphanussativus (Terras FRG et al, 1992, J Biol Chem, 267:15301-13309) andrelated proteins such as Bn-AFP1 and Bn-AFP2 from Brassica napus,Br-AFP1 and Br-AFP2 from Brassica rapa, Sa-AFP1 and Sa-AFP2 from Sinapisalba, At-AFP1 from Arabidopsis thaliana, Dm-AMP1 and Dm-AMP2 from Dahliamerckii, Cb-AMP1 and Cb-AMP2 from Cnicus benedictus, Lc-AFP fromLathyrus cicera, Ct-AMP1 and Ct-AMP2 from Clitoria ternatea(International Patent Application Publication Number WO93/05153published 18 Mar. 1993).

These publications are specifically incorporated herein by reference.

These and other plant-derived antimicrobial proteins are useful asfungicides or antibiotics to improve the disease-resistance ordisease-tolerance of crops either during the life of the plant or forpost-harvest crop protection. The proteins may be extracted from planttissue or produced by expression within micro-organisms or synthesised.Exposure of a plant pathogen to an antimicrobial protein may be achievedby application of the protein to plant parts using standard agriculturaltechniques (eg surface spraying). The proteins may also be used tocombat fungal or bacterial disease by expression within plant bodies(rather than just at the surface). The antimicrobial protein may beexpressed in an endophyte introduced into plant tissue. DNA encoding theantimicrobial proteins (which may be a cDNA clone, a genomic DNA cloneor DNA manufactured using a standard nucleic acid synthesiser) may alsobe transformed into a plant, and the proteins expressed withintransgenic plants. For example, transgenic tobacco expressing a barleyribosome inactivating protein has increased resistance to the fungalpathogen Rhizoctonia solani (Logemann et al, 1992, Biotechnol,10:305-308); transgenic tobacco expressing a barley α-thionin hasincreased resistance to Pseudomonas bacterial pathogens (Carmona et al,1993, Plant J, 3(3):457-462); transgenic tobacco expressing a beanchitinase has increased resistance to the fungal pathogen Rhizoctoniasolani (Broglie et al, 1991, Science, 254:1194-1197).

Another group of plant proteins have recently been linked to a potentialrole in plant defence. Non-specific lipid transfer proteins (hereinafterreferred to as nsLTPs) are a family of proteins of unknown function,which are classified as lipid transfer proteins based on their abilityto shuttle phospholipids between membrane vesicles or organelles invitro. These proteins are able to translocate phospholipids or otherapolar compounds between two membrane systems. Non-specific lipidtransfer proteins have been isolated from both mono- and dicotyledonousspecies, including;

Spinacia oleracea (So-nsLTP; Bernhard WR et al, 1990, Plant Physiol,95:164-170);

Ricinus communis (CB-A, CB-B and CB-C; Takishima K et al, 1988, Eur JBiochem, 190:107-112);

Daucus carota (Dc-nsLTP or EP2; Sterk et al, 1991, Plant Cell,9:907-921);

Nicotiana tabacum (TobLTP; Masuta C et al, 1992, FEBS Lett; 311:119-123);

Hordeum vulgare (PAPI, Mundy J and Rogers JC, 1986, Planta, 169:51-63));

Zea mays (Zm-nsLTP; Tchang F et al, 1988, J Biol Chem, 263:16849-16855).

These proteins were previously thought to play a role in cytoplasmiclipid shuttling between organelles, that is the transport ofphospholipids from endoplasmic reticulum to cell and organelle membranes(Arondel V and Kader JC, 1990, Experientia, 46, 579-585). However,recent evidence shows that at least some nsLTPs are locatedextra-cellularly, making their proposed function in membrane biogenesisunlikely (Sterk P et al, 1991, Plant Cell, 3, 907-921; Thoma S et al,1993, Plant J, 3:427-436).

We have previously described an antimicrobial protein isolated fromradish seeds, designated Rs-nsLTP (Raphanus sativus non-specific lipidtransfer protein) because of its homology with non-specific lipidtransfer proteins isolated from other plant species (InternationalPatent Application Publication Number WO93/05153 published on 18 Mar.1993). Rs-nsLTP inhibits the growth of several fungi in vitro and shows38 to 53% sequence identity with a variety of non-specific lipidtransport proteins from other plant sources. We have therefore proposeda model in which nsLTPs play a role in defence against microbial attack(Terras FRG et al, 1992, Plant Physiol, 100:1055-1058).

Molina A et al (1993, FEBS Letters, 316(2):119-122) isolated fourhomogeneous proteins (CW18, CW20, CW21, CW22) from barley leaves whichinhibited growth of the pathogens Clavibacter michiganensis subsp.sepedonicus, Pseudomonas solanacearum and Fusarium solani. The aminoacid sequences of these proteins were homologous to known nsLTPs fromplants (32-62% identical positions). A homologous protein (Cw₄₁) waspurified from maize leaves and also found to have inhibitory properties.Molina et al therefore proposed a defence role for non-specific lipidtransfer proteins from plants. International Patent ApplicationPublication Number WO92/20801 (Universidad Politecnica de Madrid;published on 26 Nov. 1992) discusses the antipathogenic activity(particularly antibacterial activity) of phospholipid transfer proteins(particularly the barley proteins CW18, CW20, CW21 AND CW22),antipathogenic compositions containing such proteins, DNA sequencesencoding such proteins and transgenic plants expressing such proteins.

We have now identified novel potent antimicrobial proteins with broadspectrum activity against plant pathogenic fungi and with someantibacterial activity.

According to the present invention, there is provided an antimicrobialprotein having substantially the amino acid sequence shown in SEQ ID NO1.

An antimicrobial protein according to the invention is capable ofisolation from seeds of the family Alliaceae, in particular from thegenus Allium. Such proteins may also be isolated from the seeds of bothrelated and unrelated species, or may be produced or synthesised by anysuitable method.

The invention further provides a DNA sequence encoding a proteinaccording to the invention, and a vector containing said sequence. TheDNA may be cloned or transformed into a biological system allowingexpression of the encoded protein.

In a further aspect, the invention provides plants transformed with DNAencoding an antimicrobial protein according to the invention.

The invention further provides a process of combating fungi or bacteriawhereby they are exposed to the proteins according to the invention.

An antimicrobial protein according to the invention has been isolatedfrom seeds of Allium cepa (onion) and is hereinafter called Ace-AMP1(Allium cepa--Antimicrobial Protein 1). Ace-AMP1 shows activity againsta range of plant pathogenic fungi.

The amino acid sequence of the Ace-AMP1 protein has been determined bydirect sequencing of the protein and by translation of the full-lengthAce-AMP1 cDNA sequence. Ace-AMP1 has a unique primary structure.Although it is partially homologous to non-specific lipid transferproteins (nsLTPs) from various plant sources, Ace-AMP1 is distinguishedfrom the nsLTPs in several ways. Ace-AMP1 deviates at 22% of thepositions where all known nsLTPs share conserved residues. In contrastto nsLTPs, Ace-AMP1 is extremely rich in arginine (19 arginines in 93residues; approximately 20% of amino acid content is arginine). Asdiscussed above, some nsLTPs have shown antimicrobial activity. However,the antimicrobial activity of Ace-AMP1 is considerably stronger thanthat of nsLTPs (see comparative tests in Example 7). Ace-AMP1 shows aparticularly strong antifungal activity and a particularly broadspectrum of antifungal activity. Moreover, the antimicrobial activity ofAce-AMP1 is significantly higher than that of the nsLTPs when assessedin the presence of inorganic cations at physiological concentrations. Inaddition, Ace-AMP1 appears to have no lipid transfer activity: testshave shown that, in contrast to nsLTPs like those isolated from maize orwheat seeds, Ace-AMP1 was unable to transfer phospholipids fromliposomes to mitochondria. As a further distinction, the structure ofthe cDNA clone encoding Ace-AMP1 has a preproprotein structure whereascDNA encoding known nsLTPs has a preprotein structure (see Examples 9,10 and 11).

An antimicrobial protein according to the invention is a protein havingantifungal activity and having an amino acid sequence substantially asshown in SEQ ID NO 1. In particular, an antimicrobial protein accordingto the invention is rich in arginine.

Knowledge of its primary structure enables manufacture of theantimicrobial protein, or parts thereof, by chemical synthesis using astandard peptide synthesiser. It also enables production of DNAconstructs encoding the antimicrobial protein. The DNA sequence may bepredicted from the known amino acid sequence or the sequence may beisolated from plant-derived DNA libraries.

Oligonucleotide probes may be derived from the known amino acid sequenceand used to screen a cDNA library for cDNA clones encoding some or allof the protein. These same oligonucleotide probes or cDNA clones may beused to isolate the actual antimicrobial protein gene(s) by screeninggenomic DNA libraries. Such genomic clones may include control sequencesoperating in the plant genome. Thus it is also possible to isolatepromoter sequences which may be used to drive expression of theantimicrobial (or other) proteins. These promoters may be particularlyresponsive to environmental conditions (such as the presence of a fungalpathogen), and may be used to drive expression of any target gene.

The Ace-AMP1 cDNA has been isolated using PCR-based cloning as describedin Example 9.

DNA encoding the antimicrobial protein (which may be a cDNA clone, agenomic DNA clone or DNA manufactured using a standard nucleic acidsynthesiser) can then be cloned into a biological system which allowsexpression of the protein or a part of the protein. The DNA may beplaced under the control of a constitutive or inducible promoter.Examples of inducible systems include pathogen induced expression andchemical induction. Hence the protein can be produced in a suitablemicro-organism or cultured cell, extracted and isolated for use.Suitable micro-organisms include Escherichia coli, Pseudomonas andyeast. Suitable cells include cultured insect cells and culturedmammalian cells. The genetic material can also be cloned into a virus orbacteriophage. The DNA can also be transformed by known methods into anyplant species, so that the antimicrobial protein is expressed within theplant.

Plant cells according to the invention may be transformed withconstructs of the invention according to a variety of known methods(Agrobacterium Ti plasmids, electroporation, microinjection,microprojectile gun, etc). The transformed cells may then in suitablecases be regenerated into whole plants in which the new nuclear materialis stably incorporated into the genome. Both transformedmonocotyledonous and dicotyledonous plants may be obtained in this way.

Examples of genetically modified plants which may be produced includefield crops, cereals, fruit and vegetables such as: oilseed rape,canola, sunflower, tobacco, sugarbeet, cotton, soya, maize, wheat,barley, rice, sorghum, tomatoes, mangoes, peaches, apples, pears,strawberries, bananas, melons, potatoes, carrot, lettuce, cabbage,onion.

The antimicrobial proteins of the invention show surprisingly highactivity and inhibit the growth of a variety of plant pathogenic fungiat submicromolar doses. The proteins not only show a wide range ofantifungal activity but also activity against Gram positive bacteria.The proteins are thus useful as fungicides or antibiotics, foragricultural or pharmaceutical applications. Exposure of a plantpathogen to an antimicrobial protein may be achieved by expression ofthe protein within a micro-organism (including an endophyte) which isapplied to a plant or the soil in which a plant grows. The proteins mayalso be used to combat fungal or bacterial disease by application of theprotein to plant parts using standard agricultural techniques (egspraying). An antimicrobial composition may comprise an antimicrobiallyeffective amount of the protein together with an agriculturallyacceptable carrier and/or adjuvant customarily used in agriculturalprotein formulations (including solid or liquid adjuvants, solvents,surfactants, etc). The proteins may also be used to combat fungal orbacterial disease by expression within plant bodies, either during thelife of the plant of for post-harvest crop protection. The protein mayalso be used as a fungicide or anti-bacterial to treat mammalianinfections, or for preservation of products susceptible to contaminationby micro-organisms (for example, processed food products).

The antimicrobial proteins may be isolated and purified from appropriateseeds, synthesised artificially from their known amino acid sequence, orproduced within a suitable micro-organism by expression of recombinantDNA. The proteins may also be expressed within a transgenic plant.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention may be further understood by reference to the drawings, inwhich:

FIGS. 1A and 1B show the cation exchange chromatogram for Ace-AMP1 andthe associated graph of fungal growth inhibition.

FIGS. 2A and 2B show the HPLC profile of purified Ace-AMP1.

FIGS. 3A and 3B show the alignment of the amino acid sequences ofAce-AMP1 and various plant non-specific lipid transfer proteins (SEQ IDNOS: 1-14).

FIGS. 4A and 4B show the sequences of the Ace-AMP1 cDNA (SEQ ID NO: 15)and translated protein (SEQ ID NO: 16).

FIGS. 5A and 5B are diagrams of the vectors pFAJ3033 and pFAJ3034.

The invention may also be further understood by reference to theSequence Listing, in which:

SEQ ID NOs 1 to 14 refer to the amino acid sequences in FIGS. 3A and 3B:

SEQ ID NO 1 is mature Ace-AMP1;

SEQ ID NO 2 is Rs-nsLTP;

SEQ ID NO 3 is So-nsLTP;

SEQ ID NO 4 is EP2;

SEQ ID NO 5 is TobnsLTP;

SEQ ID NO 6 is Le-nsLTP;

SEQ ID NO 7 is CB-A;

SEQ ID NO 8 is CB-B;

SEQ ID NO 9 is CB-C;

SEQ ID NO 10 is PAPI;

SEQ ID NO 11 is CW18;

SEQ ID NO 12 is CW21;

SEQ ID NO 13 is Ta-nsLTP;

SEQ ID NO 14 is Zm-nsLTP;

SEQ ID NOs 15 to 16 refer to the sequences in FIGS. 4A and 4B:

SEQ ID NO 15 is the nucleic acid sequence of the Ace-AMP1 cDNA;

SEQ ID NO 16 is the amino acid sequence of Ace-AMP1 translated from thecDNA sequence;

SEQ ID NOs 17 to 25 refer to the oligonucleotides listed in Table 5;

SEQ ID NO 17 is OWB114;

SEQ ID NO 18 is OWB116;

SEQ ID NO 19 is OWB117;

SEQ ID NO 20 is OWB111;

SEQ ID NO 21 is OWB132;

SEQ ID NO 22 is OWB133;

SEQ ID NO 23 is OWB158;

SEQ ID NO 24 is OWB159;

SEQ ID NO 25 is OWB160.

The following Examples illustrate the invention.

EXAMPLE 1

Antifungal and Antibacterial Activity Assays.

Antifungal activity was measured by microspectrophotometry as previouslydescribed (Broekaert, 1990, FEMS Microbiol Lett, 69:55-60). Routinely,tests were performed with 20 μl of a (filter-sterilized) test solutionand 80 μl of a suspension of fungal spores (2×10⁴ spores/ml) in eitherhalf strength potato dextrose broth (medium A) or half strength potatodextrose broth with CaCl₂ and KCl added to final concentrations of 1 mMand 50 mM respectively (medium B).

For experiments on the antagonistic effect of cations, a syntheticgrowth medium was used. The synthetic growth medium consisted of K₂ HPO₄(2.5 mM), MgSO₄ (50 μM), CaCl₂ (50 μM), FeSO₄ (5 μM), CoCl₂ (0.1 μM),CuSo₄ (0.1 μM), Na₂ MoO₄ (2 μM), H₃ BO₃ (0.5 μM), KI (0.1 μM), ZnSO₄(0.5 μM), MnSO₄ (0.1 μM), glucose (10 g/l), asparagine (1 g/l),methionine (20 mg/l), myo-inositol (2 mg/l), biotin (0.2 mg/l),thiamine-HCl (1 mg/l), and pyridoxine-HCl (0.2 mg/l).

Unless otherwise stated the test organism was Fusarium culmorum (strainIMI 180420) and incubation was done at 25° C. for 48 hours. Theantifungal activity of a sample (units per ml) is defined as the totalvolume of the assay mixture divided by the volume of the sample in theassay mixture that gives 50 percent growth inhibition (=dilution factorfor 50 percent growth inhibition). Percent growth inhibition is definedas 100 times the ratio of the corrected absorbance of the controlmicroculture minus the corrected absorbence of the test microcultureover the corrected absorbence at 595 nm of the control microculture. Thecorrected absorbence values equal the absorbence at 595 nm of theculture measured after 48 hours minus the absorbence at 595 nm measuredafter 30 min.

Antibacterial activity was measured microspectrophotometrically asfollows. Bacteria were pre-cultured overnight in 2% Tryptone at 30° C.in a rotary shaker. A soft agarose medium (2% tryptone; 0.5% low meltingpoint agarose) was inoculated with the bacteria to a cell density of 10⁵colony forming units/ml). Aliquots (80 μl) of the bacterial suspensionwere added to filter-sterilized samples (20 μl) in flat-bottom 96-wellmicroplates and allowed to solidify. The absorbence at 595 nm of theculture was measured with the aid of a microplate reader after 30minutes and 24 hours of incubation at 28° C. Percent growth inhibitionwas calculated as described above for the antifungal activity assay.

Antibiotic activity on yeast was determined as for the antibacterialassay, except that the growth medium consisted of half strength potatodextrose broth (Difco) and 0.5% low melting point agarose. Eighty μl ofa suspension of yeast cells in the latter medium (10⁶ cells/ml) wasadded to 20 μl of the test solution.

EXAMPLE 2

Extraction of basic heat-stable proteins from Allium cepa seeds

One hundred grammes of Allium cepa seeds (from AVEVE, Belgium) wereground in a coffee mill and the resulting meal was extracted for 2 hoursat 4° C. with 200 ml of an ice-cold extraction buffer containing 10 mMNaH₂ PO₄, 15 mM Na₂ HPO₄, 100 mm KCl, 2 mM EDTA and 2 mM thiourea. Afterextraction, the slurry was mixed in a WARING blender and subsequentlysqueezed through a jam mincer to separate the extract from the solidresidue. The resulting extract was clarified by centrifugation (10 minat 5,000× g). Solid ammonium sulphate was added to the supernatant toobtain 85% relative saturation and the precipitate allowed to form bystanding overnight at 4° C. Following centrifugation at 7,000× g for 30minutes, the precipitate was redissolved in 100 ml distilled water anddialyzed extensively against distilled water. After dialysis thesolution was adjusted to 50 mM NH₄ Ac (pH 9) by addition of the ten-foldconcentrated buffer and passed over a Q-Sepharose Fast Flow (Pharmacia,Uppsala, Sweden) column (12×5 cm) equilibrated in 50 mM NH₄ Ac (pH 9).The protein fraction which passed through the column was lyophilised andredissolved in 200 ml 50 mM NH₄ Ac (pH 5.5).

This material represents the basic (pI>9) protein fraction of the seeds.This fraction was further purified as described in Example 3.

EXAMPLE 3

Purification of an antimicrobial protein from Allium cepa seeds

The starting material for the isolation of the Allium cepa antimicrobialprotein was the basic protein fraction extracted from the mature seedsas in Example 2. Proteins were further purified by cation exchangechromatography of this extract.

Approximately 200 ml of the basic protein fraction was applied to aS-Sepharose High Performance (Pharmacia) column (10×1.6 cm) equilibratedin 50 mM NH₄ Ac, pH 5.5. The column was eluted at 2.0 ml\min with alinear gradient from 50 mM to 2M NH₄ Ac, pH 5.5 over 180 minutes. Theeluate was monitored for protein by online measurement of the absorbenceat 280 nm (results shown in FIG. 1B) and collected in 20 ml fractions.One ml samples from each fraction were dried by lyophilisation, andredissolved in 1 ml of distilled water of which 20 μl was assayed forantifungal activity as described in Example 1 (Results shown in FIG. 1A)in both medium A and B.

Following chromatography, the extract yielded a broad and complex peakof antifungal activity, composed of at least two active components withdifferent sensitivity to presence of CaCl₂ and KCl in the assay medium(Medium B) and a well-defined active peak eluting at approximately 1.5MNH₄ -acetate. The latter peak, being the less antagonised by Ca²⁺ and K⁺could be further purified by reverse-phase HPLC. One ml of this peakfraction was loaded on a PEP-S (porous silica C₂ /C₁₈, Pharmacia) column(25×0.4 cm) equilibrated with 0.1% TFA (trifluoracetic acid). The columnwas developed at 1 ml/min with a linear gradient of 0.1% TFA to 100%acetonitrile/0.1% TFA over 50 minutes. The eluate was monitored forprotein by online measurement of the absorption at 280 nm (results shownin FIG. 2B). One ml fractions were collected, vacuum dried, andredissolved in 1 ml distilled water of which 20 μl was used in anantifungal assay as described in Example 1 (results shown in FIG. 2A).The first single well-resolved peak of activity was called Ace-AMP1(Allium cepa--Antimicrobial Protein 1).

EXAMPLE 4

Molecular structure of the purified antimicrobial protein, Ace-AMP1

The molecular structure of the purified antimicrobial protein wasfurther analysed. Sodium dodecyl sulphate polyacrylamide gelelectrophoresis (SDS-PAGE) was performed on precast commercial gels(PhastGel 8-25% from Pharmacia) using a PhastSystem (Pharmacia)electrophoresis apparatus. The sample buffer contained 200 mM Tris-HCl(pH 8.3), 1% (w/v) SDS, mM EDTA, 0.005% bromophenol blue and, unlessotherwise stated, 1% (w/v) dithioerythritol (DTE). Proteins were fixedafter electrophoresis in 12.5% glutaraldehyde and silver-stainedaccording to Heukeshoven and Dernick (1985, Electrophoresis, 6:103-112).Molecular weight markers run for comparison were: phosphorylase B (97.4kDa), bovine serum albumin (66.2 kDa), ovalbumin (42.7 kDa), carbonicanhydrase (31.0 kDa), soybean trypsin-inhibition (21.5 kDa) and lysozyme(14.4 kDa).

SDS-PAGE analysis of reduced and unreduced Ace-AMP1 revealed a singleband of approximately 10 kDa and 22 kDa, respectively. The molecularweight of about 10 kDa of the reduced Ace-AMP1 could be confirmed by asimilar SDS-PAGE on PhastGel High Density (Pharmacia) which allowsincreased resolution for proteins below 20 kDa. Determination of themolecular mass of native Ace-AMP1 by gel filtration on Superose-12(Pharmacia) yielded a value of about 7.5 kDa. The SDS-PAGE molecularmass value of 22 kDa for unreduced Ace-AMP1 may be an overestimation dueto a relatively low SDS binding capacity of this compact protein.

Determination of covalently bound sugars using the phenol-sulphuric acidmethod of Dubois et al (1956, Anal Chem, 28:350-356) and D-glucose as astandard, was negative, suggesting that Ace-AMP1 is not glycosylated.

All cysteine residues of Ace-AMP1 appeared to participate in disulphidebonds, as unreduced Ace-AMP1 did not contain free thiol groups. Thiolgroup determination was done by the dithionitrobenzoic acid method ofEllman, GL (1959; Arch Biochem Biophys, 82:70-74) using 10 ml ofprotein. Reduced protein samples were prepared by reaction with 10 mMDTT for 1 hour at 45° C. followed by extensive dialysis againstdistilled water.

EXAMPLE 5

Amino acid sequencing of Ace-AMP1

Cysteine residues were modified by S-carboxyamidomethylation asdescribed in Cammue BPA et al, 1992, J Biol Chem, 267:2228-2233.Reagents were removed by HPLC on a Pep-S (porous silica C₂ /C₁₈)(Pharmacia) column (25×0.4 cm). The S-carboxyamidomethylated proteinswere recovered by eluting the column with a linear gradient from 0.1%trifluoracetic acid (TFA) to acetonitrile containing 0.1% TFA. Theresulting protein fractions were subjected to amino acid sequenceanalysis in a 477A Protein Sequence (Applied Biosystems) with on-linedetection of phenylthiohydantoin amino acid derivatives in a 120AAnalyser (Applied Biosystems).

Initial attempts to sequence Ace-AMP1 showed that the protein wasN-terminally blocked. Since deblocking with pyroglutamate aminopeptidase (Boehringer, FRG) was unsuccessful, Ace-AMP1 was digested withthe endoproteinases Arg-C and Asp-N (both of sequencing grade fromBoehringer, FRG). Digestion was done according to the manufacturers'instructions on reduced and S-carboxyamidomethylated Ace-AMP1 applyingminimal advised enzyme to protein ratios (w/w) and maximal advisedincubation times. Digested peptides were subsequently separated byRP-HPLC on a Pep-S (porous silica C₂ /C₁₈ ; Pharmacia) column (25×0.4cm) using a linear elution gradient from 0.1% TFA to acetonitrilecontaining 0.1% TFA in 100 minutes at 1 ml/minute. Digestion with Arg-Cresulted in at least 10 separable peptides, suggesting already arelatively high arginine content of Ace-AMP1. Treatment with Asp-Ngenerated 3 protein fragments. After sequencing of these peptides theprimary structure of Ace-AMP1 was reconstructed with exception of theblocked N-terminal part.

The Ace-AMP1 amino acid sequence was found to be partially homologouswith non-specific lipid transfer proteins (nsLTPs) from different plantsources, including: Rs-nsLTP from Raphanus sativus seeds (Terras FRG etal, 1992, Plant Physiology; 100: 1055-1058); So-nsLTP from Spinaciaoleraceae leaves (Bernhard WR et al, 1991, Plant Physiology, 95:164-170); EP2 from Daucus carota zygotic embryos (Sterk P et al, 1991,Plant Cell, 3:907-921); TobLTP from Nicotiana tabacum flowers (Masuta Cet al, 1992, FEBS Lett; 311:119-123); Le-nsLTP from Lycopersiconesculente (Tonnes-Schumann S et al, 1992, Plant Mol Biol, 18:749-757);CB-A, CB-B and CB-C from Ricinus communis seedlings (Takishima K et al,1988, Eur J Buiochem, 190:1070112); PAPI from Hordeum vulgare seeds(Mundy J and Rogers JC, 1986, Planta, 169:51-63); CW18 and CW21 fromHordeum vulgare leaves (Molina A et al, 1993, FEBS Lett, 316:119-122);Ta-nsLTP from Triticum aestivum (Simorre JP et al, 1991, Biochem,30:11600-11608); Zm-nsLTP from Zea mays seedlings (Tchang F et al, 198,J Biol Chem, 263: 16849-16855). A sequence comparison of Ace-AMP1 withthese nsLTPs is given in FIGS. 3A and 3B. Gaps introduced for optimalalignment are represented by dashes. The first nine N-terminal aminoacids are derived from the nucleotide sequence of Ace-AMP1 cDNA (seeExample 5).

From a comparison of the nsLTP sequences shown in FIGS. 3A and 3B (allsequences excluding Ace-AMP1), the following consensus motif can bederived. All eight cysteines are at conserved positions 4, 14, 30, 31,51, 53, 77 and 93 (numbering as in FIG. 3); hydrophobic residues (L, I,A, V, M) or aromatic residues (F, W, Y) appear at positions 2, 7, 11,17, 18, 34, 37, 41, 54, 61, 64, 69, 73, 82, 85, 87, and 96; prolines arepresent at positions 25 and 74; basic residues (H, R, K) are conservedat positions 47 and 55; hydroxy residues (S, T) appear at positions 43and 88; and a conserved aspartic acid occupies position 46. Ace-AMP1partly corresponds to this consensus motif, but deviates at thefollowing positions: it does not have hydrophobic/aromatic residues atpositions 2, 18, 61 and 69; it does not have the conserved asparticacid, lysine and serine at positions 46, 55 and 88 respectively. Hence,about 22% of the conserved residues in nsLTP proteins are altered inAce-AMP1. Moreover, Ace-AMP1 distinguishes itself from all other knownnsLTP sequences by a much higher arginine content. Ace-AMP1 contains atleast 19 arginines whereas the number of arginines in the nsLTP proteinsvaries from 1 (So-nsLTP) to 6 (Zm-nsLTP).

It is noted that most cysteine-rich antibiotic peptides found inanimals, such as defensins (Lehrer RI et al, 1991, Cell, 64:229-230),β-defensins (Selsted ME et al, 1993, J Biol Chem, 268:6641-6648) andbactenecins (Romeo D et al, 1988, J Biol Chem, 263:9573-9575; Gennaro Ret al, 1989, Infect immun, 57:3142-3146) are also particularly rich inarginine.

EXAMPLE 6

Stability of the protein's antifungal activity

Table 1 summarises the results of further testing of the stability ofthe antifungal activity of Ace-AMP1.

Tests for antifungal activity were performed with 20 μl samples dilutedfive-fold with growth medium containing Fusarium culmorum spores,according to the assay method given in Example 1. Untreated controlsamples consisted of the test proteins at 100 μg/ml in 10 mM sodiumphosphate buffer (pH 7). Heat stability tests were performed by heatingaliquots of the test proteins for 10 minutes at different temperaturesup to 100° C. For digestions, proteases were added at 400 μg/ml andincubated at 37° C. for 16 hours.

                  TABLE 1                                                         ______________________________________                                        Stability of the antifungal activity of Ace-AMP1                                               Relative antifungal                                                           activity                                                     Treatment        (% of control activity)                                      ______________________________________                                        Control          100                                                          Heating at 80° C., 10 min                                                               100                                                          Heating at 90° C., 10 min                                                               100                                                          Heating at 100° C., 10 min                                                              100                                                          Chymotrypsin digestion                                                                          80                                                          Pronase E digestion                                                                             5                                                           Proteinase K digestion                                                                          60                                                          Trypsin digestion                                                                               90                                                          ______________________________________                                    

The antifungal activity of Ace-AMP1 was not affected by heat treatmentsup to 100° C. for 10 minutes. Ace-AMP1 was relatively resistant totreatments with chymotrypsin, trypsin and proteinase K while digestionwith pronase E reduced the activity almost completely.

EXAMPLE 7

Antifungal potency of Ace-AMP1

The antifungal potency of the purified protein was assessed on differentplant pathogenic fungi, using the assay described in Example 1. Growthof fungi, collection and harvest of fungal spores were done aspreviously described (Broekaert et al, 1990, FEMS Microbiol Lett,69:55-60). The following fungal strains were used: Alternaria brassicolaMUCL 20297, Ascochyta pisi MUCL 30164, Botrytis cinerea MUCL 30158,Colletotrichum lindemuthianum MUCL 9577, Fusarium culmorum IMI 180420,Fusarium oxysporum f.sp. in IMI 236441, Fusarium oxysporum f.sp.lycopersici MUCL 909, Nectria haematococca Collection Van Etten 160-2-2,Phoma betae MUCL 9916, Pyrenophora tritici-repentis MUCL 30217,Pyricularia oryzae MUCL 30166. Verticillium dahliae MUCL 6963.

Serial dilutions of the antifungal proteins were applied to the fungi,using a synthetic growth medium for fungi (SMF) (See Example 1)supplemented with (SMF⁺) or without (SMF⁻) CaCl₂ and KCl to finalconcentrations of 1 mM and 50 mM, respectively. The percent growthinhibition was measured by microspectrophotometry. The concentrationrequired for 50% growth inhibition after 48 h of incubation (IC₅₀ value)was calculated from the dose-reponse curves.

The IC₅₀ values of Ace-AMP1 on different plant pathogenic fungi arepresented in Table 2, where they are compared with those determinedunder the same conditions for three nsLTPs, namely Rs-nsLTP (data fromTerras et al, 1992, Plant Physiol. 100:1055-1058), Zm-nsLTP and Ta-nsLTP(isolated as described in Simorre et al, 1991, Biochem, 30:11600-11608).

Both in media SMF- and SMF+, Ace-AMP1 inhibits all twelve tested fungiby 50% at concentrations equal or below 10 μg/ml (corresponding to about1 μM). Ace-AMP1 is therefore a potent plant antifungal proteinexhibiting a broad inhibitory spectrum.

It is surprising that Ace-AMP1 is almost as active in SMF⁺ as in SMF⁻.The activity of an antifungal protein in a cation containing medium suchas SMF⁺ is believed to be of physiological relevance since all plantcell compartments contain relatively high cation concentrations (TerrasFRG et al, 1992, J Biol Chem, 267:15301-15309).

The potency of Ace-AMP1 in SMF⁺ compares very favourable to otherrelatively cation-insensitive antifungal proteins such as Rs-AFP2 whichinhibits 8 out of 12 fungi listed in Table 2 at concentrations below 10μg/ml (Terras FG et al, 1992, J Biol Chem, 267:15301-15309). Ace-AMP1 isalso much more potent than a recently described nsLTP-like protein fromRaphanus sativus, Rs-nsLTP (Terras FRG et al, 1992, Plant Physiol,100:1055-1058) which is partly homologous to Ace-AMP1 (see FIGS. 3A and3B). Indeed, none of the fungi listed in Table 2 are inhibited byRs-nsLTP in SMF⁺ at concentrations below 100 μg/ml (Terras FRG et al,1992, Plant Physiol, 100:1055-1058). Moreover, two nsLTPs isolated frommaize and wheat, Zm-nsLTP and Ta-nsLTP respectively, did not inhibitgrowth of any of the nine fungi tested in SMF+ at concentrations below200 EMG/ML (see Table 2). The IC₅₀ value on Fusarium solani of nsLTPproteins isolated from barley leaves (including CW18 and CW21, see FIGS.3A and 3B) varied from approximately 25 to 180 μg/ml (depending on theisoform) when assessed in potato dextrose broth as a medium (Molina A etal, 1993, FEBS Lett, 316:119-122). However, the activity of theseproteins on other fungi and their sensitivity to cations have not beendescribed.

The activity of Ace-AMP1 on Fusarium culmorum in synthetic growth mediumsupplemented with different cations (assayed as described in Example 1)has been compared directly with the activity of the Ac-AMP1antimicrobial peptide from Amaranthus caudatus seeds (Broekaert et al,1992, Biochemistry, 31: 4308-4314) and of β-purothionin from wheatendosperm (another type of plant seed protein with antimicrobialactivity; Redman DG and Fischer N, 1969, J Sci Food Agri, 20: 427-432).Table 3 summarises the IC₅₀ values under different conditions. WhereasAc-AMP1 is very sensitive to the presence of all tested cations, theactivities of Ace-AMP1 and β-purothionin seem to be rathercation-stimulated although not by Ca²⁺. The antagonistic effect of Ca²⁺is, however, much less pronounced on Ace-AMP1 than on the thionin.

                                      TABLE 2                                     __________________________________________________________________________    Antifungal activity of Ace-AMP1, Rs-nsLTP, Zm-nsLTP and Ta-nsLTP              on different phytopathogenic fungi                                                     IC.sub.50 (μg/ml)                                                          Ace-AMP1                                                                              Rs-nsLTP Zm-nsLTP                                                                              Ta-nsLTP                                    FUNGUS   SMF-                                                                              SMF+                                                                              SMF-                                                                              SMF+ SMF-                                                                              SMF+                                                                              SMF-                                                                              SMF+                                    __________________________________________________________________________    A brassicola                                                                           2.5 1.5 48  500  >200                                                                              >200                                                                              >200                                                                              >200                                    A pisi   1.0 10.0                                                                              41  700  >200                                                                              >200                                                                              >200                                                                              >200                                    B cinerea                                                                              3.0 7.0 45  680  nd  nd  nd  nd                                      C lindemuthianum                                                                       1.5 1.5 25  >1000                                                                              >200                                                                              >200                                                                              >200                                                                              >200                                    F culmorum                                                                             6.0 10.0                                                                              20  520  200 >200                                                                              >200                                                                              >200                                    F oxysporum pisi                                                                       3.5 4.0 58  900  200 >200                                                                              >200                                                                              >200                                    F oxysporum                                                                   lycopersici                                                                            3.0 10.0                                                                              54  >1000                                                                              200 >200                                                                              >200                                                                              >200                                    N haematococca                                                                         3.5 7.0 100 >1000                                                                               60 >200                                                                              >200                                                                              >200                                    P betae  1.5 7.0 18  750  150 >200                                                                              >200                                                                              >200                                    P tritici-repentis                                                                     3.0 3.5 nd  nd   nd  nd  nd  nd                                      P orvzae 3.0 7.0 10  >1000                                                                              nd  nd  nd  nd                                      V dahliae                                                                              0.25                                                                              0.5  7  135  200 >200                                                                              >200                                                                              >200                                    __________________________________________________________________________     (nd = not determined)                                                    

                  TABLE 3                                                         ______________________________________                                        Antifungal activity of Ace-AMP1, Ac-AMP1 and                                  β-purothionin on Fusarium culmorum in synthetic                          medium supplemented with different cations                                    IC50 (μg/ml)                                                                         +50     +50     +50   +5     +5   +5                                          mM      mM      mM    mM     mM   mM                                SMF       K+      Na+     NH4+  Mg2+   Ba2+ Ca2+                              ______________________________________                                        Ace-   3      2       2     1.5   2        2    6                             AMP1                                                                          Ac-AMP1                                                                              4      100     100   50    >200   >200 >200                            β-puro-                                                                         4      2       3      2    2      2.5   35                             thionin                                                                       ______________________________________                                    

EXAMPLE 8

Anti-bacterial and anti-yeast activity of Ace-AMP1

The purified protein was assessed for its effect on the growth of thefollowing bacteria: Bacillus megaterium ATCC 13632, Sarcina lutea ATCC9342, Agrobacterium tumefaciens LMG 188, Alcaligenes eutrophus LMG 1195,Azospirillum brasilense ATCC 29145, Erwinia carotovora subsp carotovoraLMG2458, Escherichia coli strain HB101, Pseudomonas solanacearum LMG2293, Pseudomonas syringae pv tabaci LMG 5192 and Xanthomonas campestrispv campestris LMG 582. It was also assessed for its effect on the growthof Saccharomyces cerevisiae strain Sp1. Bioassays were carried out asdescribed in Example 1. The results are summarised in Table 4.

                  TABLE 4                                                         ______________________________________                                        Activity of Ace-AMP1, Rs-nsLTP, Zm-nsLTP, Ta-nsLTP                            on bacteria and yeast                                                                   IC50 (μg/ml)                                                     MICROORGANISM                                                                             Ace-AMP1  Rs-nsLTP Zm-nsLTP                                                                             Ta-nsLTP                                ______________________________________                                        B megaterium                                                                              0.8        20       60    >200                                    S lutea     8.0       >200     >200   >200                                    A tumefaciens                                                                             >200      nd       nd     nd                                      A eutrophus >200      nd       nd     nd                                      A brasilense                                                                              >200      nd       nd     nd                                      E carotovoza                                                                              >200      >200     >200   >200                                    E coli      >200      nd       nd     nd                                      P solanacearum                                                                            >200      nd       nd     nd                                      P syringae  >100      >200     >200   >200                                    X campestris                                                                              >100      >200     >200   >200                                    S cerevisiae                                                                              >200      nd       nd     nd                                      ______________________________________                                         nd = not determined                                                      

Ace-AMP1 inhibits growth of both Gram positive bacteria tested (Bmegaterium and S lutea) but has little or no effect on any of the eightdifferent Gram negative bacteria which were tested or on the yeast Scerevisiae. Rs-nsLTP and Zm-nsLTP are only inhibitory to B megaterium,but are at least 10-fold less active on this bacterium than Ace-AMP1.The ns-LTPs isolated from barley leaves (including CW18 and CW21, seeFIG. 3) have been reported to inhibit growth of the Gram positivebacterium Clavibacter michiganensis subsp sepedonicus and the Gramnegative bacterium P solanacearum (Molina et al, 1993, FEBS Lett,316:119-122).

EXAMPLE 9

PCR-based cloning of the 5' and 3' parts of Ace-AMP1 cDNA

Total RNA was extracted from a mixture of immature seeds collected 15,21 and 30 days post anthesis.

The 3' part of Ace-AMP1 cDNA was cloned as follows. Total RNA (1 μg) wasreverse transcribed in a 30 μl reaction mixture containing 12 units ofavian myeloblastosis virus reverse transcriptase (Boehringer Mannheim),appropriate buffer constituents (Sambrook et al, 1989, MolecularCloning, Cold Spring Harbor Laboratory Press) and 10 pmol of a modifiedoligo-dT primer (primer OWB114, see Table 5) and incubated for 30 min at52° C. A fraction of the reverse transcription reaction (0.5 μl) wastransferred to a 25 μl PCR reaction mixture containing 5 pmol of theantisense primer OWB114, 5 pmol of the sense primer OWB111 (adegenerated primer corresponding to an internal amino acid sequence ofAce-AMP1, namely PRFQNIP), 5 nmol dNTPs, 0.5 units of Taq polymerase andTaq polymerase buffer constituents (Sambrook et al, 1989, MolecularCloning, Cold Spring Harbor Laboratory Press). Temperature cycling forPCR was done according to standard conditions (Sambrook et al., 1989,Molecular Cloning, Cold Spring Harbor Laboratory Press) using a primerannealing temperature of 55° C. PCR reaction products were analysed byagarose gel electrophoresis and a band of about 400 bp (that was absentfrom control PCR reactions containing the same template but only one ofboth primers) was isolated using a Prep-a-Gene kit (Biorad) according tothe manufacturers instructions. The PCR product was digested with XbaI,subcloned into the plasmid pEMBL18+ (Boehringer Mannheim) and the insertsequenced on an ALF automated sequencer (Pharmacia) using the Autoreadsequencing kit (Pharmacia) with fluoresceine-labelled M13 forward andreverse primers.

The 5' part of the Ace-AMP1 cDNA was cloned as follows. Total RNA wasreverse transcribed as described above using either OWB114 or OWB133 (anAce-AMP1 specific primer, derived from the nucleotide sequence of the 3'part of Ace-AMP1 cDNA) as a primer. Excess primer was removed by gelfiltration over a Chromaspin+TE-100 (Clontech) column equilibrated in 10mM Tris, 1 mM EDTA, 300 mM NaCl, 0.05% (w/v) SDS (pH 8). RNA wassubsequently removed by alkaline hydrolysis, the ssDNA was ethanolprecipitated as described by Delort et al (1989, Nucl Acids Res,17:6439-6448), and finally redissolved in 10 μl distilled water. The 3'end of these ssDNA preparations (corresponding to the 5' end of themRNA) were ligated to the oligonucleotide OWB116 which was synthesizedwith a phosphate group at its 5' end (to allow for ligation to thessDNA) and an amino group at its 3' end (to avoid primer self-ligation).The ssDNA ligation reaction mixture (30 μl) contained 5 pmol of primerOWB116, 2.5 μl of ssDNA (see above), 10 units of T4 RNA ligase (NewEngland Biolabs) and T4 RNA ligase buffer constituents (Tessier et al,1986, Anal Biochem, 158:171-178), and incubation was done at 22° C. for16 h. A fraction (0.1 μl) of the ssDNA ligation mixture was transferredto a 25 μl PCR reaction mixture containing 5 nmol of primer OWB117(which is partially complementary to OWB116), 5 mmol of dNTPs, 1.25units of Taq polymerase and Taq polymerase buffer constituents. After 5PCR cycles with an annealing temperature of 60° C., 25 pmol of anAce-AMP1-specific primer (OWB132, corresponding to a position onAce-AMP1 cDNA immediately upstream of that of OW133) was added to thereaction mixture and 30 additional PCR cycles with an annealingtemperature of 55° C. were carried out. A PCR product of about 400 bpwhich was not present in single primer PCR controls was gel-purified asdescribed above. The same 400 bp PCR band was obtained irrespective ofwhether OWB133 or OWB114 were used in the first strand synthesis. ThisPCR product was BamHI-digested, subcloned into pEMBL18+ and thenucleotide sequence of the insert determined as described above.

By combining the nucleotide sequences of the 5' and 3' parts (whichoverlapped by 38 nucleotides) a 686 bp sequence was obtained thatcorresponds to full length Ace-nsLTP cDNA.

Ace-AMP1 cDNA contains a 396 bp open reading frame coding for 132 aminoacids, a 36 bp 5' leader sequence and a 3' untranslated region of 232 bpup to the poly (A+) tail (FIGS. 4A and 4B). Analysis of the codingregion reveals the presence of a putative signal peptide of 27 aminoacids. The predicted signal peptide cleavage site (indicated by an arrowin FIGS. 4A and 4B) is in agreement with the rules of von Heijne (1986,Nucl Acids Res, 14:4683-4690) and with the observation that most matureplant nsLTPs have a cysteine at positions 4 and a valine at position 7.The amino acid sequence between amino acids 37 and 120 of the codingregion (underlined in FIGS. 4A and 4B) identical to the amino acidsequence determined experimentally for mature Ace-AMP1. The cDNA derivedcoding region predicted that mature Ace-AMP1 has 9 additional aminoacids at the N-terminus relative to the sequence determined in Example5. This sequence could not be determined experimentally due to thepresence of a blocked N-terminal amino acid in mature Ace-AMP1.Furthermore, the translation product of Ace-AMP1 mRNA has 12 amino acidsat its carboxyl-terminus which are absent from mature Ace-AMP1. Thiscarboxyl-terminal propeptide is rich in hydrophobic and acidic residues,a characteristic feature of carboxyl-terminal propeptides present in theprecursors of vacuolar plant proteins (Nakamura and Matsuoka, 1993,Plant Physiol, 101:1-5).

Such carboxyl-terminal propeptides have in a number of cases beendemonstrated to be determinants for targetting the protein to thevacuole (Bednarek and Raikhel, 1991, Plant Cell, 3,:1195-1206; Neuhauset al, 1991, Proc Natl Acad Sci USA, 88:10362-10366). All nsLTP-likeproteins have been shown to be translated as preproteins, which deviatesfrom the preproprotein structure found in the case of Ace-AMP1 (Arondeland Kader, 1990, Experientia, 46:579-585; Madrid and von Wettstein,1991, Plant Physiol Biochem, 29:705-711).

TABLE 5

Oligonucleotides used for Ace-AMP1 cDNA cloning

Name Sequence

OWB114 (SEQ ID NO: 17) 5'-CCACTCTAGAGAATTCACCTTTTTTTTTTTTTTTTTTTT-3'

OWB116 (SEQ ID NO: 18) 5'-AGAATTCGCATTGCATCGGATCCATGATCGAT-3'

OWB117 (SEQ ID NO: 19) 5'-ATCGATCATGGATCCGATGCAATGC-3'

OWB111 (SEQ ID NO: 20) 5'-AATTCTAGACCNMGNTTYCARAAYATHCC-3'

OWB132 (SEQ ID NO: 21) 5'-ATCGGATCCGAATTCGTGTTGCGACAATCACGAGG-3'

OWB133 (SEQ ID NO: 22) 5'-ATCGGATCCGAATTCAGGACGAACAAAGGTGTTGC-3'

OWB158 (SEQ ID NO: 23) 5'-TAAGGTACCATGGTTCGCGTTGTATC-3'

OWB159 (SEQ ID NO: 24) 5'-TAAGGATCCTTCAGTTAATCCTGCCGCATTGAATTCG-3'

OWB160 (SEQ ID NO: 25) 5'-TAAGGATCCCTTCATTCCTCAGCGTCCAAG-3'

The following oligonucleotides have a sense orientation relative toAce-AMP1 mRNA: OWB117, OWB111, OWB158. The remaining oligonucleotides(OWB114, OWB116, OWB132, OWB133, OWB159, OWB160) have an antisenseorientation relative to Ace-AMP1 mRNA. The position of eacholigonucleotide relative to the Ace-AMP1 cDNA nucleotide sequence is asfollows:

OWB114 poly(A⁺) tail

OWB116 5'-end

OWB117 5'end

OWB111 307-326

OWB132 325-344

OWB133 338-354

OWB158 35-53

OWB159 372-396

OWB160 417-437.

Restriction sites in the oligonucleotides are underlined in Table 5. InOWB116, the 5'OH at the 5' end is phosphorylated and the 3'OH at the 3'end is aminated. The sequence of OWB117 is complementary to nucleotides8-32 of OWB116. In OWB111: N=G,A,T,C; H=A,C,T; M=A,C; Y=C,T; R=A,G.

EXAMPLE 10

Construction of an expression vector

Total onion seed RNA was reverse translated using primer OWB114 asdescribed in example 9. Fractions (0.5 μl) of the reaction mixture wereused in PCR amplification reactions under standard conditions (Sambrooket al, 1989, Molecular Cloning, Cold Spring Harbor Laboratory Press)using either the primer combination OWB158-OWB159 at a primer annealingtemperature of 65° C. or the primers OWB158-OWB160 at a primer annealingtemperature of 55° C. Primer OWB158 introduces a KpnI site immediatelyupstream of the natural NcoI site of Ace-AMP1 cDNA (which encompassesthe start codon), primer OWB159 introduces a stop codon and a BamHI sitebehind the codon of amino acid 120 (the last amino acid of matureAce-AMP1), and primer OWB160 introduces a BamHI site behind the naturalstop codon of Ace-AMP1. The resulting OWB158-OWB159 and OWB158-OWB160amplification products were digested with KpnI and BamHI and subclonedinto the corresponding sites of plasmid pBluescript II SK--to yieldplasmids pAce2 and pAce1, respectively. The inserts were verified bynucleotide sequencing. The inserts of plasmids pAce1 and pAce2 wereisolated by digestion with NcoI and SacI and subsequently ligated intothe corresponding sites of the expression vector pBI505 (Datla et al,1993, Plant Science, 94:139-149), thus creating plasmids pAce3 andpAce4, respectively. In the expression vector pAce3, the coding regionof Ace-AMP1 is flanked at its 5 end by the strong constitutive promoterof the 35S RNA of cauliflower mosaic virus with a duplicated enhancerelement (to allow for high transcriptional activity, Kay et al, 1987,Science, 236:1299-1302) and the 5' leader sequence of the alfalfa mosaicvirus (to allow for high translational activity, Datla et al, 1993,Plant Science, 94:139-149). The coding region of the Ace-AMP1 cDNA isflanked at its 3' end by the polyadenylation sequence of theAgrobacterium tumefaciens nopaline synthase gene (Bevan et al, 1983,Nature, 304:184-187). Vector pAce4 is identical to pAce3 except that thecoding region lacks the domain encoding the 12 carboxyl-terminal aminoacids of the propeptide.

EXAMPLE 11

Construction of plant transformation vectors

The expression vectors pAce3 and pAce4 described in example 10 weredigested with HindIII and SacI and the fragments containing the Ace-AMP1expression cassettes were subcloned into the HindIII-SacI digested planttransformation vector pGPTV-KAN (Becker et al, 1992, Plant Mol Biol,20:1195-1197) yielding plant transformation vectors pFAJ3033 andpFAJ3034, respectively. A schematic representation of these vectors isshown in FIGS. 5A and 5B.

The symbols used in FIGS. 5A and 5B are as follows:

RB: right border of T-DNA

LB: left border of T-DNA

Tnos: terminator of T-DNA nopaline synthase gene

CTPP: carboxy-terminal propeptide domain of Ace-AMP1 cDNA

MP: mature protein domain of Ace-AMP1 cDNA

SP: signal peptide domain of Ace-AMP1 cDNA

AMV: alfalfa mosaic virus 5' leader sequence

Penh35S: promoter of 35S RNA of cauliflower mosaic virus with duplicatedenhancer region

Pnos: promoter of T-DNA nopaline synthase gene

nptII: coding region of neomycin phosphotransferase II gene

Tg7: terminator of T-DNA gene 7

EXAMPLE 12

Plant Transformation

The disarmed Agrobacterium tumefaciens strain LBA4404 (pAL4404) (Hoekemaet al, 1983, Nature 303, 179-180) is transformed with the transformationvector using the method of de Framond et al (BioTechnology, 1:262-269).

Tobacco transformation is carried out using leaf discs of Nicotianatabacum Samsun based on the method of Horsch et al (1985, Science,227:1229-1231) and co-culturing with Agrobacterium strains containingpFAJ3033 or pFAJ3034. Co-cultivation is carried out under selectionpressure of 100 μg/ml kanamycin. Transgenic plants are regenerated onmedia containing 100 μg/ml kanamycin. These transgenic plants may beanalysed for expression of the newly introduced genes using standardwestern blotting techniques. Plants capable of constitutive expressionof the introduced genes may be selected and self-pollinated to giveseed. F1 seedlings of the transgenic plants may be further analysed forincreased resistance to plant pathogens.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 25                                                 (2) INFORMATION FOR SEQ ID NO: 1:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 93 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Ace-AMP1                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:                                      GlnAsnIleCysProArgValAsnArgIleValThrProCysValAla                              151015                                                                        TyrGlyLeuGlyArgAlaProIleAlaProCysCysArgAlaLeuAsn                              202530                                                                        AspLeuArgPheValAsnThrArgAsnLeuArgArgAlaAlaCysArg                              354045                                                                        CysLeuValGlyValValAsnArgAsnProGlyLeuArgArgAsnPro                              505560                                                                        ArgPheGlnAsnIleProArgAspCysArgAsnThrPheValArgPro                              65707580                                                                      PheTrpTrpArgProArgIleGlnCysGlyArgIleAsn                                       8590                                                                          (2) INFORMATION FOR SEQ ID NO: 2:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 42 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Rs-nsLTP                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:                                      AlaLeuSerCysGlyThrValAsnSerLeuAsnAlaAlaCysIleGly                              151015                                                                        TyrLeuThrGlnAsnAlaProLeuAlaArgGlyCysCysThrGlyVal                              202530                                                                        ThrAsnLeuAsnAsnMetAlaThrThrPro                                                3540                                                                          (2) INFORMATION FOR SEQ ID NO: 3:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 91 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: So-nsLTP                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:                                      GlyIleThrCysGlyMetValSerSerLysLeuAlaProCysIleGly                              151015                                                                        IleLeuLysGlyGlyProLeuGlyGlyGlyCysCysGlyGlyIleLys                              202530                                                                        AlaLeuAsnAlaAlaAlaAlaThrThrProAspArgLysThrAlaCys                              354045                                                                        AsnCysLeuLysSerAlaAlaAsnAlaIleLysGlyIleAsnTyrGly                              505560                                                                        LysAlaAlaGlyLeuProGlyMetCysGlyValHisIleProTyrAla                              65707580                                                                      IleSerProSerThrAsnCysAsnAlaValHis                                             8590                                                                          (2) INFORMATION FOR SEQ ID NO: 4:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 94 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: EP2                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:                                      ValLeuThrCysGlyGlnValThrGlyAlaLeuAlaProCysLeuGly                              151015                                                                        TyrLeuArgSerGlnValAsnValProValProLeuThrCysCysAsn                              202530                                                                        ValValArgGlyLeuAsnAsnAlaAlaArgThrThrLeuAspArgLys                              354045                                                                        ThrAlaCysGlyCysLeuLysGlnThrAlaAsnAlaValThrGlyLeu                              505560                                                                        AsnLeuAsnAlaAlaAlaGlyLeuProAlaArgCysGlyValAsnIle                              65707580                                                                      ProTyrLysIleSerProThrThrAspCysAsnArgValVal                                    8590                                                                          (2) INFORMATION FOR SEQ ID NO: 5:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 91 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: TobLTP                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:                                      AlaLeuSerCysGlyGlnValGlnSerGlyLeuAlaProCysLeuPro                              151015                                                                        TyrLeuGlnGlyArgGlyProLeuGlySerCysCysGlyGlyValLys                              202530                                                                        GlyLeuLeuGlyAlaAlaLysSerLeuSerAspArgLysThrAlaCys                              354045                                                                        IleCysLeuLysSerAlaAlaAsnAlaIleLysGlyIleAspMetGly                              505560                                                                        LysAlaAlaGlyLeuProGlyAlaCysGlyValAsnIleProTyrLys                              65707580                                                                      IleSerProSerThrAspCysSerLysValGln                                             8590                                                                          (2) INFORMATION FOR SEQ ID NO: 6:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 91 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Le-nsLTP                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:                                      AlaLeuThrCysGlyGlnValThrAlaGlyLeuAlaProCysLeuPro                              151015                                                                        TyrLeuGlnGlyArgGlyProLeuGlyGlyCysCysGlyGlyValLys                              202530                                                                        AsnLeuLeuGlySerAlaLysThrThrAlaAspArgLysThrAlaCys                              354045                                                                        ThrCysLeuLysSerAlaAlaAsnAlaIleLysGlyIleAspLeuAsn                              505560                                                                        LysAlaAlaGlyIleProSerValCysLysValAsnIleProTyrLys                              65707580                                                                      IleSerProSerThrAspCysSerThrValGln                                             8590                                                                          (2) INFORMATION FOR SEQ ID NO: 7:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 92 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: CB-A                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:                                      ValAspCysGlyGlnValAsnSerSerLeuAlaSerCysIleProPhe                              151015                                                                        LeuThrGlyGlyValAlaSerProSerAlaSerCysCysAlaGlyVal                              202530                                                                        GlnAsnLeuLysThrLeuAlaProThrSerAlaAspArgArgAlaAla                              354045                                                                        CysGluCysIleLysAlaAlaAlaAlaArgPheProThrIleLysGln                              505560                                                                        AspAlaAlaSerSerLeuProLysLysCysGlyValAspIleAsnIle                              65707580                                                                      ProIleSerLysThrThrAsnCysGlnAlaIleAsn                                          8590                                                                          (2) INFORMATION FOR SEQ ID NO: 8:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 92 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: CB-C                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:                                      ValAsnCysGlyGlnValAsnLysAlaLeuSerSerCysValProPhe                              151015                                                                        LeuThrGlyPheAspThrThrProSerLeuThrCysCysAlaGlyVal                              202530                                                                        MetLeuLeuLysArgLeuAlaProThrValLysAspLysArgIleAla                              354045                                                                        CysGluCysValLysThrAlaAlaAlaArgTyrProAsnIleArgGlu                              505560                                                                        AspAlaAlaSerSerLeuProTyrLysCysGlyValValIleAsnVal                              65707580                                                                      ProIleSerLysThrThrAsnCysHisGluIleAsn                                          8590                                                                          (2) INFORMATION FOR SEQ ID NO: 9:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 92 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: CB-B                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:                                      AlaValProCysSerThrValAspMetLysAlaAlaAlaCysValGly                              151015                                                                        PheAlaThrGlyLysAspSerLysProSerGlnAlaCysCysThrGly                              202530                                                                        LeuGlnGlnLeuAlaGlnThrValLysThrValAspAspLysLysAla                              354045                                                                        IleCysArgCysLeuLysAlaSerSerLysSerLeuGlyIleLysAsp                              505560                                                                        GlnPheLeuSerLysIleProAlaAlaCysAsnIleLysValGlyPhe                              65707580                                                                      ProValSerThrAsnThrAsnCysGluThrIleHis                                          8590                                                                          (2) INFORMATION FOR SEQ ID NO: 10:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 93 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: PAPI                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:                                     AlaLeuAsnCysGlyGlnValAspSerLysMetLysProCysLeuThr                              151015                                                                        TyrValGlnGlyGlyProGlyGlyProSerGlyLeuCysCysAsnGly                              202530                                                                        ValArgAspLeuHisAsnGlnAlaGlnSerSerGlyAspArgGlnThr                              354045                                                                        ValCysAsnCysLeuLysGlyIleAlaArgGlyIleHisAsnLeuAsn                              505560                                                                        LeuAsnAsnAlaAlaSerIleProSerLysCysAsnValAsnValPro                              65707580                                                                      TyrThrIleSerProAspIleAspCysSerArgIleTyr                                       8590                                                                          (2) INFORMATION FOR SEQ ID NO: 11:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 90 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: CW18                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:                                     AlaIleThrCysGlyGlnValSerSerAlaLeuGlyProCysAlaAla                              151015                                                                        TyrAlaLysGlySerSerThrSerProSerAlaGlyCysCysSerGly                              202530                                                                        ValLysArgLeuAlaGlyLeuAlaArgSerThrAlaAspLysGlnAla                              354045                                                                        ThrCysArgCysLeuLysSerValAlaGlyAlaTyrAsnAlaGlyArg                              505560                                                                        AlaAlaGlyIleProSerArgCysGlyValSerValProTyrThrIle                              65707580                                                                      SerAlaSerValAspCysSerLysIleHis                                                8590                                                                          (2) INFORMATION FOR SEQ ID NO: 12:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 90 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: CW21                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:                                     AlaIleSerCysGlyGlnValSerSerAlaLeuSerProCysIleSer                              151015                                                                        TyrAlaArgGlyAsnGlyAlaLysProProAlaAlaCysCysSerGly                              202530                                                                        TyrLysArgLeuAlaGlyAlaAlaGlnSerThrAlaAspLysGlnAla                              354045                                                                        ThrCysArgCysIleLysSerAlaAlaGlyGlyLeuAsnAlaGlyLys                              505560                                                                        AlaAlaGlyIleProSerMetCysGlyValSerValProTyrAlaIle                              65707580                                                                      SerAlaSerValAspCysSerLysIleArg                                                8590                                                                          (2) INFORMATION FOR SEQ ID NO: 13:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 90 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Ta-nsLTP                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:                                     IleAspCysGlyHisValAspSerLeuValArgProCysLeuSerTyr                              151015                                                                        ValGlnGlyGlyProGlyProSerGlyGlnCysCysAspGlyValLys                              202530                                                                        AsnLeuHisAsnGlnAlaArgSerGlnSerAspArgGlnSerAlaCys                              354045                                                                        AsnCysLeuLysGlyIleAlaArgGlyIleHisAsnLeuAsnGluAsp                              505560                                                                        AsnAlaArgSerIleProProLysCysGlyValAsnLeuProTyrThr                              65707580                                                                      IleSerLeuAsnIleAspCysSerArgVal                                                8590                                                                          (2) INFORMATION FOR SEQ ID NO: 14:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 93 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Zm-nsLTP                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:                                     AlaIleSerCysGlyGlnValAlaSerAlaIleAlaProCysIleSer                              151015                                                                        TyrAlaArgGlyGlnGlySerGlyProSerAlaGlyCysCysSerGly                              202530                                                                        ValArgSerLeuAsnAsnAlaAlaArgThrThrAlaAspArgArgAla                              354045                                                                        AlaCysAsnCysLeuLysAsnAlaAlaAlaGlyValSerGlyLeuAsn                              505560                                                                        AlaGlyAsnAlaAlaSerIleProSerLysCysGlyValSerIlePro                              65707580                                                                      TyrThrIleSerThrSerThrAspCysSerArgValAsn                                       8590                                                                          (2) INFORMATION FOR SEQ ID NO: 15:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 686 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (111MOLECULETYPE:cDNA                                                         (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Ace-AMPl                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:                                     AACGAAAATTACGAAATTACATCAATATCTCGAGCCATGGTTCGCGTTGTATCTTTACTT60                GCAGCATCGACCTTCATACTGTTGATTATGATAATCAGCAGTCCGTATGCARATAGTCAG120               AACATATGCCCAAGGGTTAATCGAATTGTGACACCCTGTGTGGCCTACGGACTCGGAAGG180               GCACCAATCGCCCCATGCTGCAGAGCCCTGAACGATCTACGGTTTGTGAATACTAGAAAC240               CTACGACGTGCTGCATGCCGCTGCCTCGTAGGGGTAGTGAACCGGAACCCCGGTCTGAGA300               CGAAACCCTAGATTTCAGAACATTCCTCGTGATTGTCGCAACACCTTTGTTCGTCCCTTC360               TGGTGGCGTCCAAGAATTCAATGCGGCAGGATTAACCTTACGGATAAGCTTATATACTTG420               GACGCTGAGGAATGAAGACTAGGCTCTACTGTTATGCACTATAGTTTATAGTATATATAC480               TAAATAAAACAGTATGTGCTGTATAATTTGCAATATGGACTTATTTATAGCAAGTCCTAA540               TGGTGTCTGCTACTTGGGTCCAGCATTGAGCACTATATAGGCACTATATAGGGTACTATG600               GGCTGATTATGATGTCAACGGCGGTACTTTATCTTACATAAATAAATAATGGGTTTATCT660               TGCTTGAAAAAAAAAAAAAAAAAAAA686                                                 (2) INFORMATION FOR SEQ ID NO: 16:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 132 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Ace-AMP1 (translated)                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:                                     MetValArgValValSerLeuLeuAlaAlaSerThrPheIleLeuLeu                              151015                                                                        IleMetIleIleSerSerProTyrAlaAsnSerGlnAsnIleCysPro                              202530                                                                        ArgValAsnArgIleValThrProCysValAlaTyrGlyLeuGlyArg                              354045                                                                        AlaProIleAlaProCysCysArgAlaLeuAsnAspLeuArgPheVal                              505560                                                                        AsnThrArgAsnLeuArgArgAlaAlaCysArgCysLeuValGlyVal                              65707580                                                                      ValAsnArgAsnProGlyLeuArgArgAsnProArgPheGlnAsnIle                              859095                                                                        ProArgAspCysArgAsnThrPheValArgProPheTrpTrpArgPro                              100105110                                                                     ArgIleGlnCysGlyArgIleAsnLeuThrAspLysLeuIleTyrLeu                              115120125                                                                     AspAlaGluGlu                                                                  130                                                                           (2) INFORMATION FOR SEQ ID NO: 17:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: OWB114                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:                                     CCACTCTAGAGAATTCACCTTTTTTTTTTTTTTTTTTTT39                                     (2) INFORMATION FOR SEQ ID NO: 18:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: OWB116                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:                                     AGAATTCGCATTGCATCGGATCCATGATCGAT32                                            (2) INFORMATION FOR SEQ ID NO: 19:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: OWB117                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:                                     ATCGATCATGGATCCGATGCAATGC25                                                   (2) INFORMATION FOR SEQ ID NO: 20:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: OWBlll                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:                                     AATTCTAGACCNMGNTTYCARAAYATHCC29                                               (2) INFORMATION FOR SEQ ID NO: 21:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: CUNA                                                      (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: OWB132                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:                                     ATCGGATCCGAATTCGTGTTGCGACAATCACGAGG35                                         (2) INFORMATION FOR SEQ ID NO: 22:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: OWB133                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:                                     ATCGGATCCGAATTCAGGACGAACARAGGTGTTGC35                                         (2) INFORMATION FOR SEQ ID NO: 23:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: OWB158                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                      TAAGGTACCATGGTTCGCGTTGTATC26                                                  (2) INFORMATION FOR SEQ ID NO: 24:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: OWB159                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:                                     TAAGGATCCTTCAGTTAATCCTGCCGCATTGAATTCG37                                       (2) INFORMATION FOR SEQ ID NO: 25:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: OWB160                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25:                                     TAAGGATCCCTTCATTCCTCAGCGTCCAAG30                                              __________________________________________________________________________

We claim:
 1. An antimicrobial protein comprising the amino acid sequenceof SEQ. ID. NO:
 1. 2. The antimicrobial protein of claim 1 consisting ofthe amino acid sequence of SEQ. ID. NO:
 16. 3. The antimicrobial proteinof either claim 1 or claim 2 which is isolated from seeds of the familyAlliaceae.
 4. The antimicrobial protein of claim 3 which is isolatedfrom seeds of the genus Allium.
 5. The antimicrobial protein of claim 4which is the protein Ace-AMP1.
 6. An isolated nucleic acid encoding theantimicrobial protein of either claim 1 or
 2. 7. The isolated nucleicacid of claim 6 wherein the sequence of the nucleic acid comprises SEQ.ID. NO:
 15. 8. A biological system transformed with the isolated nucleicacid of claim
 6. 9. The transformed biological system of claim 8 whichis a microorganism.
 10. The transformed biological system of claim 8which is a plant.
 11. A method of inhibiting the growth of fungi orbacteria comprising exposing said fungi or bacteria to the antimicrobialprotein of either claim 1 or 2.